Interview with Holly A. Ingraham, PhD Ingraham is UCSF associate professor of physiology and obstetrics, gynecology, and reproductive sciences at UC-San Francisco. She studies the development of endocrine and neuroendocrine organs using molecular biology and mouse genetics.

In general, why do you think people are so interested in the field of sex and gender?

In sex and gender, it's a basic question and it's a fundamental question for all species, for the propagation of species, and it's at the core of who we are. So I think people are just interested.

How about you personally? What do you find interesting?

I think I'm attracted to it because it is such a basic question in biology. And it's sexy, but it's not sexy from a monetary point of view in terms of research. It is a very fundamental question and usually scientists are attracted to fundamental questions.

Overall, what would you say that we do understand and what do we still not understand in this area?

I think that in the past ten years, especially with the identification of the sex-determining gene on the Y, or the testis determining factor (SRY), we've begun to really understand the proactive development of male sexual development or male sexual differentiation. So we've begun to understand how that's initiated and some of the downstream targets, although we can't connect up all the dots. What we don't understand is how that process is kept off in the female. So we really have very few clues about that.

Explain what you mean by "kept off."

[In] the process of sex determination in vertebrates, you have this proactive program where [there is] a coordinated event of different hormones coming on to allow proper male sexual development. That has to stay off in females. Many components will be used by the male and the female, but at different times. So in the male, those components are used very early before birth-and then after birth those same components are used by the female. So it's a timing issue. In females, all of that has to stay off. And all of those hormones have to stay off because if they went on, it would be a disaster for the species. So the one hormone for instance, that we work on is called mullerian inhibitory substance, which causes the destruction of the whole female reproductive system. If it went on in females, we would basically fail to propagate as a species. So it's very important that that timing event occurs correctly.

What are scientists in this field of biology still trying to figure out?

What we still really don't understand is how the events that occur early in males are kept off in the female. That has to happen in order for there to be this bifurcation between males and females. The other thing we don't understand is if there are active processes for female development. We can understand that there needs to be a repression of the male program in females, but we don't yet know if there are really active events that cause the female to develop.

The issue of how do you define a male or a female came to a head with the issue of testing for sex in the Olympics. Can you talk about this?

As I recollect, [the Olympic committee decided to] basically look for the presence of SRY. With the discovery of SRY, they could use that rather than other tests. The problem with that is there are intersex individuals who would have a perfectly normal SRY but be phenotypically female. And the question is should they be competing as women or as men because they have this SRY. I think that it's a difficult issue. I think that if a person is androgen insensitive and looks female they should be allowed to compete as a female. But the argument could be made that early in the embryo there are some events that occur due to the presence of SRY that we don't understand, that would give an advantage of male versus female. It's just assumed that males compete better than females. Although maybe not in things like ice skating or gymnastics. I personally think females do better in that sport.

For the individuals that are intersex, where it's not clear-this gets into the problem of what is male and what is female and where is the cutoff. The human genetic pool has shown that there's a continuum between male and female, and [the Olympic Committee] has the hard decision of deciding where the cutoff is. I wouldn't want to be on that committee making those decisions. So statistically I just don't know if they have enough evidence that a person who is androgen insensitive for instance would always win and have an advantage over a normal XX female. So as a scientist, I think I couldn't make a decision without that information.

Can you briefly explain androgen insensitivity syndrome?

Androgen insensitivity syndrome is usually a mutation in the androgen receptor. The androgen receptor is present all over the place and it basically responds to the hormone androgen, or testosterone. What happens in androgen insensitivity is you have mutations in a hormone nuclear receptor. [Normally], when a hormone binds to a receptor, you get these really neat conformational changes that then cause gene expression to ensue. Most androgen insensitivity cases involve mutations where the hormone would be binding. Because of those mutations, none of those conformational events occur and you don't have the downstream events occurring. None of the genes that would be turned on normally by androgen are turned on, so none of the proper things that occur in terms of male sexual development [occur] in those individuals with androgen insensitivity.

It sounds like it is more complicated that just not being able to detect testosterone.

Well, the most common mutations are in the receptor. But actually, in those individuals, because they're just insensitive to androgen, they also make things like MIS, which causes the destruction of the female reproductive system. So those individuals basically have destroyed their female reproductive system. Because they don't have testosterone, they don't have a male reproductive system, and they basically have nothing inside except for the small undeveloped gonads. They look female phenotypically, though.

Some of our most famous movie female actresses are androgen insensitive. The whole idea is that the endocrine system always works as a feed forward feed back mechanism. Because it's sensing that there's no androgen around because this receptor is mutated, you tend to produce more [androgen] and all of that excess androgen gets converted to estrogen. So they are very voluptuous; they usually have large breasts. So [AIS] individuals could have an advantage from a female point of view. In other words, every female would love to in a way be an AIS individual because they usually have large breasts and very small hips. But you can't reproduce.

What was one of the most significant discoveries in sex determination research?

[Researchers] always knew that hormones were important. What was amazing was the quest for the testis determining gene that was started relatively late in the 1950s. There were some false starts, certainly in terms of people putting forth a candidate that didn't really turn out to be right. But I think it took about 40 years and they found the right gene.

[The quest to] decide what was important on the Y chromosome really came from the work of Alfred Jost in the early '50s. His experiments really showed that there were hormones that were made by the testis, that the testis was important for getting maleness, and that there were different hormones that were made. Of course everybody knew there was an XX for female and an XY for male, and it just became a matter of time before people started looking on the Y chromosome to find out what was so important about the Y chromosome to make those hormones and cause the whole male program to follow.

Can you explain the role that intersex people have played in our understanding of sex determining genes?

The intersex patients have really given us the most information in terms of the genes that contribute to sexual differentiation and sex determination. And surprisingly enough, the mouse model, which reproduces and has all the same genes, has been more difficult to use. It's really the patients that have come into the clinic with these intersex phenotypes that have yielded the most information in terms of finding the genes, the autosomal gene specifically, that help contribute to this whole program of sex determination. Clearly it was known that the Y chromosome contributed, but of course there are many autosomal genes that also contribute to this program. Those intersex patients came in and probably didn't have any mutation in the Y chromosome that they could find or a mutation in SRY. So the search began for autosomal genes that might be downstream of SRY that would give you the same phenotype as an XY individual who was missing their SRY.

Can you explain why some people say that human development is "female by default"?

The default idea has been around in part because of the experiments that Alfred Jost did, which is to say that if you take a rabbit embryo and you cut out the ovaries, nothing much changes phenotypically. So those female bunnies still look female. But if you take out the testis very early on, then what would be a male bunny looks like a female bunny. So that's really the whole way this notion of the default pathway came up: That you need something to be proactive for male development, but you don't need anything to develop female. And that's true. If you look at intersex phenotypes or mutations in humans, there's many more individuals that are XY female than are XX male. So XX males are much rarer than XY females. Again, it's the notion that anytime you mess up the male development you become female, whereas you're female even if you might have messed up genes, you're just phenotypically female.

But now you and other researchers are working on the idea of active parts of female development, correct?

That's a hard question because figuring out what are the active components of female development is still a very difficult question. And that's because we understand what's required for males to develop, so we can start tearing apart those pathways and figuring out the genes [for male hormones, for instance]. It's much harder because we don't really know what the counterpart female hormones are. It's a much harder question to ask. People are working on it, but [they] are working on [trying] to understand what is repressed in the female. The question is, what are the active processes in females, and that's a much more difficult question. I wish I could understand it because I am female and I would like to know why I'm female and what are the active components to my gender assignment.

What are some of the genes that people are studying that might be involved in female development?

For female development, people got pretty excited in 1994 with the discovery of this gene called DAX 1, and that's in part because individuals with a duplication of that gene, or a presumed duplication of that gene, were XY females. So even in the presence of a perfectly good SRY, two copies of this gene caused them to be female. Everybody was super excited about this because this was proposed to be the ovarian-determining gene.

Over the next seven years, people [have] worked very hard to show that that was the ovarian determining gene. It was a beautiful hypothesis that really is still controversial. [It's] because as hard as people have tried, they haven't really been able to show that that's the case. In fact, there's unpublished work that suggests that it's a testis-determining gene, rather than ovary determination. So, it's really disappointing because it was such a great candidate. It could be complicated, it could be that dosage is very important; it's just very controversial and we still don't know all the answers.

There's [another] gene that has been identified by serendipity via a mouse knockout, which is called WNT 4. [This] clearly has great prospects in being one of these genes that represses the male program. It would be a great candidate to repress the male program in females. But in terms of a gene that causes ovary determination, I think we still don't have one and so people will continue to search.

Obviously male determination has a lot more to it than the SRY gene: can you talk about research into the determination pathway?

In terms of mammalian sex determination, I think there were preconceived notions that there would be this nice linear pathway starting out with SRY, and that SRY would then turn on something or turn off something and that would then turn on the next gene, and there would be this very simple cascade. What genetics has told us and molecular biology and biochemistry as well is that it's complicated. It's a web of factors that are working together. The dosage of these factors turns out to be very important in human sex determination. It's something that we should have appreciated from invertebrate sex determination pathways, but until people actually got evidence it was appreciated. Now we really know that dosage is important. We have all these downstream genes that we know interact with another, there's cross talk, cross regulation. There are probably about five or six of these genes now and there will probably be many more because this process is complicated. You want to have more than one factor that determines this process; you don't want to leave such an important event only up to one factor. You want to have checks and balances.

Clearly for male sex determination it's initiated by SRY. But oddly enough we still don't understand how SRY is talking to the rest of these factors at a real molecular or structural level. We really need to get that first leap from SRY to these other factors. We've started to fill in all the arrows with these other factors, but we don't yet know how SRY is really directing these other factors to cause male sex determination.

Can you talk about what you're doing in your lab?

We've been working on one receptor that's involved in the coordinate regulation of three hormones that are made in males in order to get proper male development. These hormones basically cause males to retain and develop their internal plumbing. Another hormone gets rid of the female plumbing. And then the third hormone allows for the testes to descend. All of that has to happen early in development in order for males to be fully functional and ready to go and reproduce when they hit puberty. You have to have the coordinated events of those hormones. There might be others, but those are the three major hormones that are needed.

The receptor that we've been working on regulates those, it's a factor that regulates the gene expression of those hormones. What we've been working on recently is to try to understand how this receptor is really working at a structural level. It's similar to the androgen receptor in that it's a nuclear receptor. The big question is: is it activated by a hormone itself and structurally, when it's activated, what does it look like. We're really reducing the question down to the smallest atom, and trying to understand at an anatomical level the structure of this receptor and how it's activated and how is it doing its job during this process of male sexual differentiation.

Talk a little bit about repressors and activators.

In terms of a biological process, in order to get it going, one always usually thinks of activators. A gene gets expressed in a particular cell type and then it activates another gene, which then activates another gene. So clearly in the sex determination pathway we have a lot of activators. The question if SRY is an activator or a repressor is still up in the air. And that's a great question and because we really don't know what SRY is doing downstream we can't answer that question yet. Everybody wants to say it activates another gene, but it could easily repress a gene. And there are other autosomal genes in the sex determination cascade further down that clearly repress genes. The gene product of WNT4 is repressing an individual from making the male steroids. That turns out to be really important in female development. You don't want to have male hormones on in females. Repression in that case turns out to be just as important as activation. Something like SOX9, which is downstream of SRY, is probably an activator that activates genes that are required for male sexual differentiation. It could still be that SRY, which is upstream, represses other genes but activates SOX9, so it could go either way, SRY could be a repressor or an activator.

But clearly we have examples where you have both activation and repression in this process. And that's common to all biological processes. The whole concept that you need both activation and repression is a common theme throughout biology.

So if it turned out that SRY is a repressor, wouldn't that challenge the female by default theory?

No, because you would say that the presence of the Y chromosome allows you to have SRY and SRY is simply repressing something that may be on in females.

You have this differential because [there is a] Y chromosome. Now, what's sort of mind boggling to think about is that in fish there aren't any sex chromosomes. So there are all these other downstream autosomal genes that are shared in common with mammals, but there's no SRY. At least with SRY or the Y chromosome you can say, well, yes, in males you have this unique chromosome, you have unique protein and that's how you set the whole thing in motion. But in fish that's a lot harder--or in alligators that are temperature sensitive, they change their sex in response to temperature. It is mind-boggling to figure out, how do you initiate this process that's important for propagation of the species without something that's different to start out with? They still don't know and it just tells you that maybe there will be other ways to initiate the male versus female process in fish that doesn't require a sex chromosome.

What are still the big unanswered questions in sex determination,?

In terms of the big unanswered questions, just thinking really small, the one thing that everybody in the field would love to know is what this gene SRY is doing at a mechanistic level. We know it's important, it's been shown to be important genetically. But for those of us who like to understand mechanism and really to understand what it's doing and how it's affecting this whole process, we really would like to know what it's doing at a molecular level. And that has been very illusive. I'm sure that the people who discovered SRY are frustrated. Many people have tried to work on this gene and the gene product, the protein of SRY, to understand what it's doing. There are many theories, none of which have really risen to the top in terms of what it's actually doing. That's a big unanswered question.

The second big unanswered question is are there proactive genes for female development, as we've already discussed. That is still a very, very large unanswered question with no candidate at this point.

The next big unanswered question is how all of these downstream autosomal genes fit together and how they interact and who's talking to who and what sort of order they come in. That's an interesting question and somebody needs to work those things out.

The greatest unanswered question for me is why the dosage of these different genes is so important in human sex determination and seems to be unimportant in mice. Why is it that factors that if you're a human being and you [have] a mutation in one allele, so your other allele is fine, and yet [you] are phenotypically the wrong sex for [your] genetic sex. That to me is a very fascinating question, why dosage is important in humans. And then when people have tried to actually reproduce or recapitulate that in mice, they seem to be completely impervious to dose. So you can have a totally screwed up allele and one normal allele, and you have a totally perfect mouse, which is not at all what you see in humans. So that's one of the big challenges, is trying to figure out what's going on in humans. And of course you can't do these genetic manipulations in humans like you can in mice.

How have we evolved in our understanding of sex determination since the 1950s?

We have now identified many of the genes that we think are important for this process. We have begun to start to understand how these genes work together. I think there's been an appreciation, especially with things like SRY, that this is a rapidly evolving process. If you look at different [species'] SRYs, what's most striking for this gene is that it's so different in different mammalian species, which is surprising. The mouse SRY is actually quite different from the human SRY except within a conserved region. That's a bit surprising given its role, its importance in male sex determination. With all of this information have come new questions, but we have a much better understanding from a genetic and molecular view about what's going on.

Can you comment on the Joan/John case: what was its impact on the field of gender identity?

I think the accounting of that story has been really interesting from the point of view of the standard medical practice, that you could basically change the phenotypic sex of a child surgically and as long as you provided hormones, [he or she] would have the appropriate gender identity. And so for Joan who became John, this is really saying that something very early in utero happens from other genes that don't make hormones. Because obviously this individual was given the appropriate hormones and yet at age 16 did not feel like they were in the right body. This suggests that there are other factors in addition to hormones that contribute to the phenotypic sex fitting with the gender identity.

For the John/Joan case, what has really challenged is this idea that it's simply hormones that are needed for gender identity to be commensurate with the phenotypic sex. So your gonadal and your reproductive plumbing are one thing, but then whether you feel like you're a girl or a boy is another thing. It was assumed that you could simply make that happen via hormones, and I think this case really shows that it's not so simple, that yes, hormones are important, but there are probably other things that are important. Maybe other hormones that are important or other gene products that are important in order for there to be a match with the phenotypic sex and the gender identity.

The other component besides hormones would be the environment, whether you gave Johnny a bunch of dolls and dressed him in pink. And in the 1970s that was really popular. Nobody wanted to acknowledge that gender really existed, that you could take a child and give a doll or a gun or a truck and that they would grow up to be completely gender unbiased. I think [the] John/Joan [case] suggests that that's not true. It probably is compatible with what most parents know if they have sons or daughters, that [a] son really wants to play with trains and trucks, and when [you] gave him a doll to play with he just simply [throws] it aside and has no interest. This was a great case to come to light, and a challenging case. It challenges us as scientists to try to figure out what is going on with gender identity.

Do you think we will figure it out?

For many of us who have been studying the molecular biology or the genetics of sex determination, this is one area that we're all extremely fascinated with. Are there genes that contribute to gender identity? Are there genes that will be shared in common that contribute to say the plumbing and the gonadal development, will they also be working in the brain to contribute to gender identity? The gene that we work on happens to be expressed in both and that's why we've gotten very interested in the question of gender identity and could the gene that we're working on help contribute to things like gender identity.

If one's going to study gender identity, you could think about using mice, but then you'd have to ask yourself, how are you going to find a transsexual mouse? Are you going to ask him? I don't even know the right questions to ask. But to be fair, there are scientists who are really tackling the question of mouse behavior and they're becoming more and more sophisticated in how they look at the behavior of mice. For instance, feeding behavior. But it's much more difficult to think about how you would ask, does this mouse have the appropriate sexual response or identity for their phenotypic sex? We as scientists will have to think harder and longer about how we might do that in mice. It's much easier to think about how you might do this in humans because you have transsexuals who are willing to undergo a huge hassle in order to change their sex. For those individuals, they clearly feel that their gender identity is not compatible with their phenotypic sex. It's much easier to ask what might be the genes that are different in those individuals that would contribute to such a strong innate feeling that they have the wrong phenotypic sex.

You can really understand how you might look at this in humans. Doing it in mice is going to be much more difficult.

What do we know about the genetics of homosexuality?

Homosexuality is an altogether different question. It is somewhat outside of the area of sex determination and gender identity because it would be sexual preference. Sexual preference is a whole other issue. It's a much more difficult issue to answer. I don't think that we have really good insights into it yet. There definitely are some scientists who have been working on describing different regions in the hypothalamus or in the brain that might be different for homosexuals versus heterosexuals. But we have a long way to go on that. It's just a politically charged question, much more so than thinking about gender identity. Gender identity is something that all of us deal with every day because we either feel like we're men or women. Sexual preference is a whole other issue that is not well understood.

Do you think we will understand it?

There are some brave individuals who will continue to work on it and try to get at the genetic nature of this difference in preference. I think that they should continue to work on it. We really don't know much about homosexuality and a change in gender preference at this point.

It's hard to know where the studies will go and being in San Francisco, I'm really aware of the homosexual community. I think many of them would like to know that there is a biological reason for the way they are.

What's your big dream?

It would be wonderful to begin to identify the genes that are important for gender identity. I think that's a fascinating question. To be able to link genes to a complex behavior is really the goal for many scientists and the behavior that I'm interested in would be how one knows if they're male or female.

How would you say the world of biology is changing in relation to sex and gender?

The [mapping] of the genome may help immensely in terms of our studies. Certainly for the identification of new genes I think it will be great. Especially if a patient comes in [and] they don't have mutations in some of the known genes, clearly knowing the human genome is going to make that much easier to identify a new gene that may contribute to this process. What the human genome won't answer is really the questions of how all of these gene products are working. Because all of these gene products make proteins and it's really those proteins that are executing the whole genetic program. There are some great new technologies, proteomics, for instance, that may [provide more answers.] With the convergence of genomics and proteomics, the field will move forward, just as every other field in biology is moving forward.

Is there anything you would want high school biology teachers to know about sex and gender?

Obviously, everybody thinks about sex. I get emails every day telling me how I could improve my sex life. Really, on a daily basis, all this junk mail. It's definitely on the minds of hormonally challenged teenagers, as we like to call them in the endocrinology field. So in teaching about sex determination and gender identity, you don't have any problem captivating a young audience and teaching them about sex because they will listen because of the word sex. But if you then get them hooked on the subject and start going through some of the beautiful genetics that have been done in this system, the molecular biology and the biochemistry that's followed from the genetics, then you can really captivate a young audience to really show them how modern biology has evolved. You can get them hooked on biology and hooked on science, which has to be the goal for all of us older scientists, to try to recruit more young scientists into the field. We continually need to recruit young scientists with new ideas in terms of thinking about areas. So the field of sex determination and gender identity is a great way to hook some of these young people into the idea of modern biology and research.